L-asparaginase (L-asparagine amidohydrolase) catalyzes the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid and ammonia. Interest in this enzyme arose a few decades ago when it was discovered that the antilymphoma activity of whole guinea pig serum was due to the enzyme L-asparaginase. Most normal tissues synthesize L-asparagine in amounts sufficient for their metabolic needs. Certain neoplastic tissues, in particular, acute lymphoblastic leukemic cells, require an exogenous source of this amino acid. L-asparaginase, by enzymatically catalyzing the hydrolysis of asparagine to aspartic acid and ammonia, deprives these malignant cells of the asparagine available from extracellular fluid, resulting in cellular death. L-asparaginase produced from Escherichia coli has a tumor inhibitory effect and the enzyme isolated from Erwinia chrysanthemi is also pharmacologically active. Since the L-asparaginases from E. coli and Erwinia chrysanthemi possess different immunological specificities, the availability of both provides an important alternative therapy if a patient happens to develop hypersensitivity to one of the enzymes (a common phenomena associated with the administration of L-asparaginase).
The process used by the only known manufacturer includes alkali lysis, multiple ion-exchange adsorptions, ammonium sulfate precipitation, multiple dialysis-concentration steps, and ethanol crystallization [Buck, et al., J. Gen. Microbiol., Vol. 65, page i (1971)]. The present invention is an improved process to produce Erwinia L-asparaginase in gram quantities for therapeutic use.
As shown in U.S. Pat. No. 3,511,754 Berk, et al, and U.S. Pat. No. 3,511,755 Ratych, et al, the use of acetone in the production of E. coli-derived L-asparaginase is known to practitioners of the art. Part of the difficulty of large-scale production of this enzyme lies in the purification process. The only major manufacturer of asparaginase purifies the enzyme by a process which includes alkali lysis, multiple ion-exchange adsorptions, ammonium sulfate precipitation, multiple dialysis-concentration steps, and ethanol crystallization [Buck, et al, J. Gen. Microbiol., Vol. 65, page i (1971)]. On the other hand, the present invention discloses a method by which significant purification of asparaginase may be achieved by initial ion-exchange chromatography. The material eluted from the ion exchange column (the starting material for the affinity column) contains only one extra protein contaminant as compared to the product available in the market. This extra protein contaminant is successfully removed by affinity chromatography. Moreover, the same purification can be achieved using affinity chromatography by itself.
Furthermore, compared to the multiple purification and dialysis steps used by Buck, et al, the process of the present invention includes only one or two chromatographic steps and one dialysis step. The enzyme is eluted from the ion-exchange column with a change in the buffer pH rather than a change in ionic strength. The enzyme coming off the column is very concentrated and contains low salt, thus avoiding a dialysis-concentration step between the ion-exchange and affinity chromatographic steps.